The field of the invention is modulation of signal transduction using casein kinase I, polynucleotides encoding casein kinase I (CKI), and variants and fragments of CKI or the polynucleotides. The invention is specifically directed to modulating the Wnt pathway using CKI polypeptides or polynucleotides. The invention is particularly directed to using CKI polypeptides or polynucleotides for diagnosis and treatment of disorders mediated by the Wnt signal pathway, especially hyperproliferative disorders, and particularly breast and colon cancer. The invention further relates to assays for screening drugs using the polypeptides and polynucleotides. The invention further relates to methods for producing the polypeptides or polynucleotides, especially by recombinant means. The invention finally relates to pharmaceutical compositions containing CKI polypeptides, polynucleotides, antibodies, variants, and fragments.
CKI is a ubiquitous protein kinase that was first described as one of the two protein kinases responsible for the Ser/Thr protein kinase activity on acidic rather than basic polypeptides in total cell extracts (Matsumara, Biochem. Biophys. Acta 289:237-241, (1972)). Since then, CKI homologs have been identified in eukaryotes from yeast to human. Several isoforms are known. Most organisms contain more than one isoform. In vertebrates seven CKI isoforms have been reported (xcex1, xcex2, xcex31, xcex32, xcex33, xcex4and xcex5). They range in size from 34 to 49 kDa (Fish et al., J. Biol. Chem. 270:14875-83 (1995); Graves et al., J. Biol. Chem. 268:6394-6401 (1993); Rowles et al., Proc. Natl. Acad. Sci. USA 88:9548-9552 (1991); Zhai et al., Biochem. Biophys. Res. Comm. 189:944-949 (1992)).
Wnt Signaling
Signaling proteins and the hierarchies in which they operate are highly conserved in evolution. This is particularly true of Wnt signaling.
The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in various developmental processes. Cell fate, morphogenesis, and mitogenesis can be affected by changes in Wnt expression. Signal transduction by the Wnt family of ligands has been recently reviewed (McMahon, Trends in Genetics 8:236-242 (1992); Nusse, et al Cell. 69:1073-1087 (1992); Dale, Biochem. J. 329:209-223 (1998)). Wnt signaling has also been addressed in relationship to the formation and function of Spemann""s organizer (Ann. Rev. Cell Dev. Biol. 13:611-667(1997)).
Drosophila gene wingless (wg) is the ortholog of the mouse Wnt-1 gene (Nusse et al., Cell 69:1073-1087 (1992); Rijsewijk et al., Cell 50:649-657 (1987)). Wg binds to its receptor frizzled on the signal receiving cell. This is believed to result in a signal that is transmitted through the disheveled (dsh) gene product (Klingensmith et al., Genes Dev. 8:118-130 (1994); Noordermeer et al., Nature 367:80-83 (1994); Theisen et al., Development 121:347-360 (1994)), ultimately resulting in regulation of the zeste white 3 (zw3) serine/threonine kinase (also known as shaggy (sgg)) (Bourouis et al., EMBO J. 9:2877-2884 (1990); Siegfried et al., Nature 367:76-80 (1994); Siegfried et al., Cell 71:1167-1179 (1992)). Zw3 in turn negatively regulates the protein levels of the armadillo (arm) gene product (Peifer et al., Dev. Biol. 166:543-556 (1994a); Peifer et al., Development 111:1029-1043 (1991); Peifer et al., Development 120:369-380 (1994b); Riggleman et al., Cell 63:549-560 (1990)). All of these proteins have vertebrate counterparts. The Dsh ortholog in Xenopus is referred to as Xdsh and in mouse as Dvl-1 (Sussman et al., Dev. Biol. 166:71-86 (1994)). The Sgg ortholog in mammals is GSK3 (Sutherland et al., Biochem. J. 296:15 (1993)). The Arm ortholog in mammals is xcex2-catenin (Peifer et al., J. Cell. Biol. 118:681-691 (1992)). Recent biochemical studies indicate that the vertebrate HMG-domain proteins Lef-1 and Xtcf-3 can physically interact with xcex2-catenin and then regulate transcription of target genes (Behrens et al., Nature 382:638-42 (1996); Molenaar et al., Cell 86:391-399 (1996)). Genetic studies indicate that pangolin (Pan), a Drosophila homolog of the mammalian Lef-1, binds to Arm protein in vivo (Brunner et al., Nature 385:829-33 (1997)). Recently, CKI was shown to associate with and phosphorylate Dsh in Drosophila (Willert et al., EMBO J. 16:3089-3096 (1997)).
Wnt binds to its receptor (a Frizzled ortholog; see below) on the cell surface. This activates Dvl-1 (Disheveled in Drosophila). Activation of disheveled inhibits GSK3 (Sgg in Drosophila) activity. Normally, GSK3 is active and phosphorylates xcex2-catenin. Phosphorylated xcex2-catenin is degraded. When GSK3 activity is inhibited, the unphosphorylated xcex2-catenin level increases, the protein enters the nucleus, binds to Lef-1 and the binary complex activates the Lef-1 enhancer causing transcription of target genes.
The Wnt signaling pathway is involved in mammary tumor and colon cancer. Ectopically-expressed Wnt-1 in mammary epithelium can induce hyperplasia, presumably by interfering with hormone-regulated Wnt pathway of other Wnt family members (Weber-Hall et al., Differentiation 57:205-214 (1994); Wong et al., Mol. Cell. Biol. 14:6278-6286 (1994)). xcex2-catenin, a component in the Wnt signal pathway, is found associated with adenomatous polyposis coli (APC) which is a familial predisposition to colon cancer (Rubinfeld et al., Science 262:1731-1734 (1993); Su et al., Science 262:1734-1737 (1993)), and the levels of free xcex2-catenin is regulated by APC together with GSK3 (Rubinfeld et al., Science 272:1023-6 (1996)). xcex2-catenin is identified as an accomplice in causing colon cancer and is strongly implicated in melanoma (Rubinfeld et al., Science 275:1790-1792 (1997)).
The Wnt-1 proto-oncogene was originally identified as a common integration site of mouse mammary tumor virus in independently isolated adenocarcinomas of mammary epithelial tissue (Nusse et al. Cell. 31:99-109 (1982)). Ectopic expression of the normally silent Wnt-1 locus results from the introduction of transcriptional enhancers contained in the mouse mammary tumor virus long terminal repeats (Nusse et al. Nature 307:131-136 (1984); Nusse et al. Cell. 31:99-109 (1982)). Formal proof of a causative role for Wnt-1 in mammary oncogenesis has come from experiments on gene transfer into mammary epithelial cell lines (Brown et al. Cell 46:1001-1009 (1986); Rijsewijk et al. EMBO J. 6:127-131 (1987)) and transgenic mice (Tsukamoto et al. Cell 55:619-625 (1988)).
Accordingly, there is a need in the art for agents that can be used to modulate the Wnt pathway and to detect disorders mediated by this pathway.
The invention is based on the inventor""s discovery that CKI can modulate the Wnt pathway. The inventor has found that normally, CKI allows a basal level of transduction in the Wnt pathway, but under-expression of CKI or a CKI variant lacking kinase activity can down-regulate the pathway, and over-expression of CKI can upregulate the pathway. A CKI variant causing over-phosphorylation should increase Wnt signal transduction. Increased signal transduction can result in a Wnt signal transduction mediated disorder, and particularly a hyperproliferative disorder. Hence, CKI or its variants or fragments can be used to modulate the Wnt pathway.
The invention encompasses the entire genus of CKI as well all species that retain the ability to affect Wnt signaling. These include homologs and orthologs from other animals or tissues as well as all isoforms.
The invention therefore provides a pharmaceutical composition comprising The invention also provides a pharmaceutical composition containing nucleic acid molecules encoding CKI.
The invention also provides variant CKI polypeptides containing a mutation in the kinase region that results in over- or under-phosphorylation of the CKI or its substrate.
The invention also provides variant CKI nucleic acid sequences containing a mutation in the kinase region that results in over- or under-phosphorylation of the CKI or its substrate.
In preferred embodiments, the CKI variant exhibits lower kinase activity than the wild-type CKI. Preferred variants contain less than approximately 50% of the activity.
In other preferred embodiments, the CKI variant has a lower capability of being phosphorylated than the wild-type CKI. In preferred embodiments, the variant CKI has less than 50% of the capability of being phosphorylated.
A specific disclosed embodiment is shown in SEQ ID NO:1, but in which amino acid 38 contains a substitution of arginine for lysine in the kinase domain (amino acids 1-69).
The invention is also directed to a CKI variant having a C-terminal deletion in the area homologous to 304-end in SEQ ID NO:1.
The invention also provides fragments of the CKI polypeptides and variants, particularly fragments containing the kinase region.
The invention also provides fragments of the CKI nucleic acid sequence and variants, particularly fragments containing the kinase region.
The invention also provides antisense nucleic acid molecules that bind to the coding strand of CKI nucleic acid molecules, particularly the kinase region.
The invention also provides ribozymes that specifically recognize and can cleave CKI nucleic acid molecules, particularly in the kinase region.
The invention also provides antibodies that selectively bind to CKI polypeptides, variants, and fragments and particularly to the kinase region.
The invention is also directed to pharmaceutical compositions containing the CKI variants, ribozymes capable of cleaving CKI MRNA, antisense polynucleotides capable of hybridizing to CKI nucleic acid, CKI antibodies, CKI non-antibody binding partners such as Dvl-1, GSK3, xcex2-catenin, and Axin, and other CKI modulators.
The invention also provides vectors and host cells for expression of the CKI nucleic acid molecules, variants, and fragments and CKI polypeptides, variants, and fragments, and particularly recombinant vectors and host cells. The invention also provides pharmaceutical compositions containing the vectors and host cells that are useful in vivo to target cells in which Wnt signaling is to be disrupted.
The invention also provides methods for making the vectors and host cells and methods for using them to produce the CKI nucleic acid molecules and polypeptides and variants and fragments.
The invention also provides methods of screening for compounds that modulate the activity of the Wnt signal pathway by means of interaction with CKI. Accordingly, these compounds can modulate the activity of the CKI polypeptide directly or can modulate the expression of CKI nucleic acid encoding the CKI polypeptide.
The invention also provides a process for modulating CKI polypeptide activity or nucleic acid expression, particularly using the screened compounds, preferably to treat disorders mediated by Wnt signal transduction.
The invention thus provides a method for interfering with the Wnt signal pathway in a cell, especially in which the pathway is up-regulated, the method comprising administering to the cell the CKI polypeptides or nucleic acids and allowing the polypeptides or nucleic acids to interfere with the pathway.
The invention also provides a method for interfering with the Wnt signal pathway in vivo in a subject, especially in which the Wnt signal pathway is up-regulated, the method comprising administering to the subject any of the CKI polypeptides or nucleic acids described herein in amounts sufficient to interfere with the pathway.
The invention also provides diagnostic assays for determining the level of CKI polypeptides or nucleic acids in a biological sample or for determining the presence of a mutation in the CKI polypeptides or nucleic acids.
The invention also provides a method for detecting a CKI-mediated hyperproliferative disorder involving isolating a sample from a patient, tissue, or cell expressing the disorder, providing a molecule capable of binding to and forming a complex with CKI, contacting the CKI sample with the molecule under conditions allowing a complex to be formed, determining the amount of complex formed, and comparing the amount of complex formed with the amount of complex formed from a normal patient, tissue, or cell.
The invention is also directed to such methods in which the disorder is a Wnt-1 signal transduction mediated disorder.
In preferred embodiments, the molecule capable of binding to CKI is an anti-CKI antibody. In alternative embodiments of the invention, the binding molecule includes, but is not limited to, GSK3, Axin, xcex2-catenin, and Dvl-1.
The invention is also directed to a method for detecting a CKI-mediated hyperproliferative disorder by obtaining a sample from a patient, tissue, or cell, expressing the disorder, contacting the sample with a CKI substrate capable of being phosphorylated by CKI, contacting the components under conditions that allow CKI phosphorylation of the substrate, and measuring the amount of phosphorylated substrate compared to a sample from a patient, tissue, or cell, not expressing the disorder.
In preferred embodiments of the invention, the substrate includes, but is not limited to, GSK3, Axin, xcex2-catenin, and Dvl-1 gene product.
The invention is also directed to a method for detecting a Wnt-1 signal transduction-mediated disorder using the above method.
The invention is also directed to a method for detecting a CKI-mediated hyperproliferative disorder by identifying specific CKI variants that lead to hyperproliferation by providing a sample from a patient, tissue, or cell expressing the disorder and detecting the variation in the variant. Detection can be by means of specific antibodies developed against the variant, peptide analysis such as by proteolytic digestion and separation, altered binding properties to CKI binding partners, and nucleic acid analysis. Nucleic acid analysis involves DNA and RNA sequencing and genomic copy analysis.
The invention is also directed to a method for detecting a Wnt-1 signal transduction-mediated disorder using the above method.
The invention is also directed to a method for detecting a CKI-mediated hyperproliferative disorder by providing a polynucleotide capable of binding to CKI nucleic acid under stringent conditions, providing a sample from a patient, tissue, or cell expressing the disorder, contacting the sample with the polynucleotide under conditions permitting a hybrid to be formed between the polynucleotide and the CKI nucleic acid, determining the amount of hybrid formed and comparing this amount with the amount of hybrid formed from a normal tissue sample.
The invention is also directed to a method for detecting a Wnt-1 signal transduction-mediated disorder by the above method.
In preferred embodiments of the method, the polynucleotide is bound to CKI mRNA.
The invention is also directed to a method of detecting a CKI-mediated hyperproliferative disorder by providing a sample from a patient, tissue, or cell expressing the disorder, contacting the sample with a protein capable of phosphorylating CKI as a substrate, contacting the sample and protein under conditions permitting the protein to phosphorylate CKI and measuring the amount of phosphorylated CKI compared to the amount of phosphorylated CKI in a normal sample.
The invention is also directed to a method for detecting a Wnt-1 signal transduction mediated disorder using the above method.
The above method allows the detection of a CKI variant that is aberrantly phosphorylated and, as a result, leads to the disorder.
The invention is also directed to a method for treating a CKI-mediated hyperproliferative disorder by administering, to an animal with the disorder, an amount of CKI inhibitor effective to treat hyperproliferation.
The invention is also directed to a method for treating a Wnt-1 signal transduction mediated disorder in an animal by administering to the animal an amount of CKI inhibitor effective to treat the disorder.
The CKI inhibitor can include a kinase inhibitor, ribozyme capable of cleaving CKI mRNA, an anti-CKI antibody, and an antisense polynucleotide capable of hybridizing to CKI mRNA to effectively inhibit translation.
The invention is also directed to a method for treating a CKI-mediated hyperproliferative disorder by administering a CKI variant in an amount effective to decrease hyperproliferation.
The invention is also directed to a method for treating a Wnt-1 signal transduction mediated disorder by administering a CKI variant in an amount effective to treat the disorder.
Preferred variants include those that exhibit less kinase activity than CKI from patients, tissues, or cells not exhibiting the disorder, or which cannot be phosphorylated to the same level as that found in CKI from patients, tissues or cells not expressing the disorder.
The invention is directed to inhibiting hyperplasia, in one embodiment by inhibiting the Wnt pathway by means of CKI. In preferred embodiments, the hyperplasia results in a tumor. In highly preferred embodiments, the tumor is a malignant tumor. In highly preferred embodiments, the tumor is a breast or colon cancer or melanoma.
The invention encompasses the treatment and diagnosis of mammals and particularly of humans.
The invention encompasses all CKI isoforms and variants from any biological source including mammals, and particularly humans. Preferred is CKIxcex5, especially as shown in SEQ ID NO:2.
Further, chimeric CKI variants can be provided in which the kinase or other functional region can be fused to heterologous CKI sequences, such as regions derived from other CKI isoforms from the same animal or other animals.